Potato Yellow Mosaic Virus Research Articles

Expression of the Oligomerization Domain of the Replication-associated Protein (Rep) of Tomato Leaf Curl New Delhi VirusInterferes with DNA Accumulation of Heterologous Geminiviruses

The minimal DNA binding domain of the replication-associated protein (Rep) of Tomato leaf curl New Delhi virus was determined by electrophoretic mobility gel shift analysis and co-purification assays. DNA binding activity maps to amino acids 1-160 (Rep-(1-160)) of the Rep protein and overlaps with the protein oligomerization domain. Transient expression of Rep protein (Rep-(1-160)) was found to inhibit homologous viral DNA accumulation by 70-86% in tobacco protoplasts and in Nicotiana benthamiana plants. The results obtained showed that expression of N-terminal sequences of Rep protein could efficiently interfere with DNA binding and oligomerization activities during virus infection. Surprisingly, this protein reduced accumulation of the African cassava mosaic virus, Pepper huasteco yellow vein virus and Potato yellow mosaic virus by 22-48%. electrophoretic mobility shift assays and co-purification studies showed that Rep-(1-160) did not bind with high affinity in vitro to the corresponding common region sequences of heterologous geminiviruses. However, Rep-(1-160) formed oligomers with the Rep proteins of the other geminiviruses. These data suggest that the regulation of virus accumulation may involve binding of the Rep to target DNA sequences and to the other Rep molecules during virus replication.

Biological and Molecular Properties of a Begomovirus from Dicliptera sexangularis

ABSTRACT Sixangle foldwing, Dicliptera sexangularis (Acanthaceae), showing severe yellow mottle and leaf distortion symptoms was collected from the shoreline of Calusa Island (Lee County, FL). The putative virus was transmitted from infected D. sexangularis to healthy seedlings by mechanical, whitefly (Bemisia tabaci biotype B), and graft-inoculations. Different forms of geminivirus-like DNAs were detected in total DNA extracted from infected plants by Southern blot hybridization analyses using DNA-A and -B of Bean golden mosaic virus (BGMV) from Guatemala as probes. Preliminary polymerase chain reaction experiments and sequence comparisons indicated that the virus was a distinct bipartite begomovirus. The virus was designated Dicliptera yellow mottle virus (DiYMV). Replicative dsDNAs of DiYMV were extracted, digested with selected restriction enzymes, and cloned into a plasmid vector. Both DNA-A and -B were sequenced and compared with those of other begomoviruses. Phylogenetic analyses using AV1, AC1, and BV1 nucleotide sequences indicated that DiYMV has a close relationship with the New World begomoviruses, especially those distributed in the nearby geographic areas of the Florida coast and the Caribbean Basin. However, different percent nucleotide sequence identities and phylogenetic relationships were detected when different open reading frames (ORFs) of DiYMV were compared with their counterparts from begomoviruses from the Caribbean Basin. Based on phylogenetic analyses of the AC1 and BV1 ORFs, DiYMV was closely related to BGMV type II isolates, whereas sequence comparisons of the common region and the AC4-derived amino acid sequences indicated its close relationship with Potato yellow mosaic virus from Venezuela.

Molecular characterization of a new whitefly-transmissible bipartite geminivirus infecting tomato in Panama.

The nucleotide sequence of infectious clones of a tomato-infecting geminivirus from Panama [named tomato leaf curl virus (ToLCV-Pan) because of symptoms produced in infected tomato (plant stunting and mild leaf curling)] was determined. ToLCV-Pan has a bipartite genome (DNAs A and B) and computer analysis showed that the genome resembles that of other bipartite, whitefly-transmitted geminiviruses. DNA A (2584 nt) and B (2542 nt) have little sequence homology other than within the common region. ToLCV-Pan clones were introduced into Lycopersicon esculentum and infected plants developed the same symptoms as naturally infected tomatoes. Homology analysis of DNA A and B showed that ToLCV-Pan is most closely related to potato yellow mosaic virus (PYMV) from Venezuela. Pseudorecombination between ToLCV-Pan and PYMV did not give viable pseudorecombinant viruses. However, in some plants infected with the pseudorecombinant virus produced by ToLCV-Pan DNA A and PYMV DNA B, systemic movement of ToLCV-Pan DNA A was observed.

Occurrence of a Strain of Potato Yellow Mosaic Geminivirus Infecting Tomato in the Eastern Caribbean.

Viruslike symptoms were observed in epidemic proportions in tomato, Lycopersicon esculentum Mill., in Guadeloupe and Martinique each year since 1992 and 1993, respectively, and in Puerto Rico since 1994 (1,3). Many tomato fields in Guadeloupe and Martinique had more than 70% of plants expressing symptoms of chlorotic mottling, leaf distortion, leaf rolling, and stunting in 1995 and 1996. The B biotype of Bemisia tabaci (aka B. argentifolii) was associated with all these epidemics. Ninety-three samples of tomato were collected from multiple locations from each island (65 samples from Guadeloupe, 11 from Martinique, and 17 from Puerto Rico) and assayed for the presence of geminivirus by polymerase chain reaction (PCR) with broad spectrum primers, PAL1v1978 and PAR1c496 for the A component, and PBL1v2040 and PCRc154 for the B component (4). Most samples tested positive for geminivirus (98% from Guadeloupe, 100% from Martinique, and 82% from Puerto Rico). Restriction analyses of amplified A component fragment with SacI, EcoRI, and AluI, and amplified B component fragment with EcoRI, AluI, VspI, PstI, and HaeIII were conducted on 34 samples (25 from Guadeloupe, six from Martinique, and three from Puerto Rico). All samples produced very similar restriction patterns, suggesting that they were infected by the same virus. One to three clones of amplified fragments of A and B components, obtained from one plant sample from each location, were at least 98.7% identical in sequence to each other and were 89.6% and 75.2 to 75.7% identical to equivalent regions in potato yellow mosaic virus (PYMV) A and B DNA, respectively (GenBank accession nos. D00940 and D00941). This isolate of PYMV was collected from potato in Venezuela in 1985 (2). Infectious full-length genomic clones of A and B component DNA from Guadeloupe were derived from the same tissue as the PCR-generated clones, and nucleotide sequences were found to be 99.1 and 99% identical to the PCR-generated fragments of A and B DNA, respectively. Nucleotide sequences of these full-length clones were 92.6 and 89.4% identical to full-length sequences of PYMV A and B DNA, respectively. The DNA sequence identity of the common regions of the A and B components between the Guadeloupe virus and PYMV was 95.1 and 91.6%, respectively. There was a nucleotide identity of 93% in the first 125 nucleotides of the coat protein gene. The virus found in Guadeloupe, Martinique, and Puerto Rico appears to be a strain of PYMV reported from Venezuela. This strain of PYMV is at least partially responsible for the epidemics in tomato in Guadeloupe, Martinique, and Puerto Rico. The similarity among geminivirus sequences at distant locations (Puerto Rico to Martinique is approximately 600 km) is unexpected and could be due to recent introductions.

Genetic Diversity Among Geminiviruses Associated with the Weed Species Sida spp., Macroptilium lathyroides, and Wissadula amplissima from Jamaica.

Genetic diversity among geminiviruses associated with three common weeds in Jamaica was studied using digoxigenin-labeled geminiviral DNA probes, polymerase chain reaction with degenerate primers for DNA-A and DNA-B, nucleic acid sequencing, and derived amino acid sequences. Geminiviruses with bipartite genomes were found in Sida spp., Macroptilium lathyroides, and Wissadula amplissima. The geminiviruses detected in Sida spp. and M. lathyroides were nearly identical and were both designated Sida golden mosaic geminivirus (SidGMV-JA), whereas the geminivirus in W. amplissima was sufficiently different to be designated Wissadula golden mosaic geminivirus (WGMV). Nucleotide sequence comparisons of the common regions and the N-terminal regions of the AC1 (rep) and AV1 ORFs, together with the derived amino acid sequence comparisons of the N-terminal parts of BC1 and BV1 ORFs were used to determine their similarities to other geminiviruses. SidGMV-JA was most similar to potato yellow mosaic geminivirus (PYMV). We propose that these two geminiviruses (SidGMV-JA and PYMV) define a new geminivirus cluster, the potato yellow mosaic virus (PYMV) cluster. WGMV was most similar to members of the Abutilon mosaic virus cluster but is not likely to be included in the Abutilon phylogenetic group because of the divergent sequence of the common region. These results indicate that geminiviruses infecting some weeds in Jamaica are distinct from crop-infecting geminiviruses in Jamaica and define a new geminivirus cluster.

Taino Tomato Mottle Virus, a New Bipartite Geminivirus from Cuba.

Geminiviruses have become the most important virus group affecting tomatoes (Lycopersicon lycopersicum (L.) Karsten) in Cuba since they have been detected in all tomato-producing areas, causing serious losses. Recently, a whitefly-transmitted, monopartite geminivirus was detected in Cuba and identified as tomato yellow leaf curl virus-Israel (TYLCV-Is) (1). Samples collected from the main tomato-producing areas during the period 1995 to1996 were further analyzed by polymerase chain reaction (PCR) with degenerate primers (PAL1v1978 and PAR1c496) (2). Whereas in samples from most areas only TYLCV was detected, in some samples from the Havana area, two DNA fragments (approximately 1.4 and 1.1 kb) were amplified by PCR. The larger fragment was identified as part of the TYLCV-Is genome, confirming the previous report (1). The 1.1-kb fragment was cloned and its nucleotide sequence suggested that a new bipartite geminivirus was also present in those tomato samples. To clone the entire genome, tomato plants were inoculated by biolistics with DNA extract from field samples. After symptom expression, a viral DNA-enriched preparation from the inoculated tomatoes was independently digested with several restriction enzymes and the products were ligated into pZero plasmid (Invitrogen, San Diego, CA). Several clones in the 2.6-kb size range were characterized by restriction mapping and hybridization against component A and B heterologous probes. Two clones were selected as containing putative A and B components and their infectivity was tested by biolistic inoculation of tomato and pepper plants. The inoculated tomatoes developed a mild mottle in the younger leaves, whereas no symptoms were visible on the inoculated pepper plants. However, the presence of viral DNA was confirmed in both tomatoes and peppers by Southern blot hybridization analysis with A- and B-specific probes. Partial sequences of both components were obtained and their analysis showed that both components shared a 170-bases common region with a 95% identity. In addition, the nucleotide sequences of two open reading frames, one in each component (AC1 and BC1), were determined and compared with geminivirus sequences deposited in Gen-Bank. A dendogram generated with the CLUSTAL program and obtained with the AC1 and BC1 amino acid sequences, placed the new geminivirus in a cluster with tomato mottle virus (ToMoV; accession nos. L14460, L14461), Abutilon mosaic virus (AbMV; X15983, X15984), potato yellow mosaic virus (PYMV; D00940, D00941), and bean dwarf mosaic virus (BDMV; M88179, M88180). The percentages of identity obtained with the amino acid sequences were as follows. For AC1: ToMoV, 87%; PYMV, 79.5%; BDMV, 78.7%; and AbMV, 78%. For BC1 protein: BDMV, 92.8%; ToMoV, 89.1%; PYMV, 88.1%; and AbMV, 67.5%. In addition, the sequences were compared with partial nucleotide sequences (AC1, coat protein [CP], and common region) of a bipartite geminivirus affecting tomatoes in Jamaica (accession nos. U83855, U83854, and U83850). Interestingly, the common regions showed a higher percentage of identity (88%) than the CP and AC1 partial nucleotide sequences (86 and 74%, respectively). These data suggest that the virus reported here is a new geminivirus and the first bipartite geminivirus reported in Cuba. Thus, the name of Taino tomato mottle virus is proposed. (Taino refers to the name of the inhabitants of Cuba at the time of Columbus's arrival in the Caribbean).

Partial Characterization of Two Whitefly-Transmitted Geminiviruses Infecting Tomatoes in Venezuela.

Whitefly-transmitted geminiviruses can cause significant yield losses on tomatoes (Lycopersicon esculentum Mill.) in Venezuela. To identify the geminivirus(es) infecting tomatoes in Venezuela, 20 tomato samples from commercial tomato fields in four states and one weed (Euphorbia heterophylla L.) sample were examined for geminivirus infection. All samples showed symptoms generally associated with geminivirus infection, including golden or yellow mosaic, mottling, crumpling and/or distortion of leaves, and, in some cases, stunted and distorted growth. Through the use of squash blot hybridization analysis and a general probe for Western Hemisphere whitefly-transmitted geminiviruses (4), geminivirus nucleic acids were detected in 19 of 20 tomato samples and the weed sample. No samples were infected with tomato yellow leaf curl virus (TYLCV), based on squash blot hybridization analysis with a TYLCV-specific probe. With polymerase chain reaction (PCR) and degenerate primers for whitefly-transmitted geminiviruses (PAL1v1978 and PAR1c496) (4), an approximately 1.2-kb DNA-A fragment was amplified from the 19 squash blot-positive tomato samples and from the weed sample. No DNA fragment was amplified from any samples when TYLCV-specific primers (PTYC1v2406 and PTYIRc287) (3) were used. PCR-amplified DNA-A fragments from four samples representing four different states [Monagas (5L), Guarico (3M), Aragua (3R), and Portuguesa (2U)] were cloned and sequenced. Partial AC1, AV1, and complete common region (CR) sequences of the 5L, 3M, and 2U DNA-A fragments were 92 to 93, 93, and 95 to 97% identical, respectively, indicating that these were DNA-A clones of the same virus. Furthermore, these sequences were 91 to 92, 92 to 95, and 93 to 95% identical, respectively, to sequences of homologous regions of potato yellow mosaic virus (PYMV), indicating that these tomato-infecting geminiviruses are isolates or strains of PYMV. The partial AC1, AV1, and complete CR sequences of the 3R DNA-A fragment were 79, 95, and 77% identical to those of 5L, 3M, and 2U clones, respectively, suggesting that this is a different geminivirus. These sequences were 75 to 87, 82 to 88, and 73 to 81% identical, respectively, to sequences of homologous regions of other tomato geminiviruses, including tomato golden mosaic from Brazil, tomato mottle from Florida, and tomato leaf crumple from Mexico. The bipartite nature of the geminiviruses that were present in the 5L, 3M, 3R, and 2U samples was suggested by the amplification of a DNA-B fragment with degenerate DNA-B primers (PBL1v2040 and PCRc1) (4). These results suggest at least two distinct bipartite Western Hemisphere whitefly-transmitted geminiviruses are associated with tomato virus diseases in Venezuela, and that one of these (sample 3R) may be an undescribed geminivirus. The sequence of the DNA-A fragment from the weed sample was not closely related to the tomato-infecting geminiviruses and, therefore, this weed was not an alternate host of these viruses. Furthermore, because PYMV has been shown to infect tomatoes and cause yellow mosaic symptoms (1), it would be of interest to determine the relationship of PYMV and tomato yellow mosaic geminivirus (ToYMV), which has been reported infecting tomatoes in Venezuela (2), but has not been characterized on the molecular level.

Complete Nucleotide Sequence of a Nonvector-Transmissible Strain of Abutilon Mosaic Geminivirus in Hawaii

The complete nucleotide sequence of a geminivirus infecting lantern 'ilima (Abutilon hybridum) in Hawaii was determined. Sequence analysis showed that this geminivirus was a strain of Abutilon mosaic geminivirus (AbMV-HI). The genome of AbMV-HI consisted of two circular, single-stranded DNA molecules of 2,634 bases (DNA-A) and 2,571 bases (DNA-B). AbMV-HI shared 95 and 91% nucleotide sequence identity with the DNA-A and DNA-B components, respectively, of a West Indian strain of AbMV (AbMV-WI). AbMV-HI also shared 90% identity with the deduced amino acid sequences of the coat proteins of tomato mottle virus, bean dwarf mosaic virus, and potato yellow mosaic virus. Results of transmission experiments showed that AbMV-HI was not transmissible by the sweet potato whitefly Bemisia tabaci. The derived amino acid sequences of the coat proteins of two nonwhitefly-transmissible strains of AbMV were compared with those of 19 whitefly-transmissible geminiviruses. Five amino acids in the N-terminal region of the coat proteins were identical for two strains of AbMV, but were different from vector-transmissible geminiviruses. These results suggested that some or all of the five amino acids at the N-terminal end of the coat protein may have been involved in whitefly transmission of bipartite geminiviruses.

Genome affinities and epitope profiles of whitefly‐transmitted geminiviruses from the Americas

SummaryThe relationships among fifteen isolates of whitefly‐transmitted geminiviruses (WTGs) from North, Central and South America and six from other continents were assessed (a) in nucleic acid hybridisation tests with sulphonated DNA probes for eight of the viruses, and/or (b) in triple‐antibody‐sandwich ELISA with panels of monoclonal antibodies (MAbs) to particles of African cassava mosaic virus (ACMV) and Indian cassava mosaic virus (ICMV). Probes specific for DNA‐A of four American viruses, abutilon mosaic (AbMV), bean golden mosaic (BGMV), squash leaf curl (SLCV) and tomato golden mosaic (TGMV), detected virtually all the American viruses but reacted weakly if at all with ICMV, ACMV or tomato yellow leaf curl virus from Thailand (TYLCV‐T). Conversely, the probe for ACMV DNA‐A did not detect any of the American viruses, and that for TYLCV‐T DNA‐A reacted weakly with SLCV and TGMV0020but did not detect the others. In contrast, probes specific for DNA‐B of the four American viruses or ACMV detected only the homologous virus, except for slight reactions between the AbMV DNA‐B probe and both chino del tomate virus (CdTV)‐DNA and SLCV‐DNA. However, a probe for DNA‐B of bean calico mosaic virus (BCMoV) reacted weakly with BGMV‐PR DNA, and a probe for DNA‐B of CdTV from Mexico detected several American viruses.Six out of 17 MAbs specific for ACMV and six out of 10 MAbs specific for ICMV reacted with one or other of the 14 American virus isolates tested. Two and‐ACMV MAbs reacted with all, and one anti‐ACMV MAb and two anti‐ICMV MAbs reacted with nearly all the American viruses, one anti‐ACMV MAb reacted with about half the American viruses and six other MAbs reacted with only one or two of them. Of the American viruses, CdTV and AbMV were the least closely related to the others. The epitope profiles of BCMoV, BGMV, cotton leaf crumple virus, serrano golden mosaic virus and SLCV were virtually indistinguishable. TGMV, potato yellow mosaic virus (PYMV) and an euphorbia virus had profiles intermediate between those of the BGMV cluster and AbMV‐CdTV. In general, the epitope profiles and the results of hybridisation tests with DNA‐A probes show that the similarities among the American viruses are greater than those between the American viruses and the viruses from other continents; the hybridisation tests with DNA‐B probes show that substantial differences exist between individual American viruses. In America, geminivirus evolution seems to have proceeded convergently from different progenitor viruses, or divergently from one ancestral form, with DNA‐B diverging to a greater extent than DNA‐A and its particle‐protein gene.

The nucleotide sequence of the infectious cloned DNA components of potato yellow mosaic virus

The complete nucleotide sequence of a Venezuelan isolate of potato yellow mosaic virus (PYMV) has been determined, showing it to be typical of subgroup I geminiviruses in that it is whitefly-transmitted, has a circular, bipartite ssDNA genome and possesses bidirectionally orientated open reading frames (ORFs). The two genomic components have little sequence similarity apart from a common region of 268 nucleotides (nt) which is almost identical. Analysis of ORFs revealed six potential coding regions encoding proteins of Mr greater than 10K, four in PYMV A (2593 nt) and two in PYMV B (2547 nt), which are preceded by regulatory transcription elements and have polyadenylation signals present at the ends. Amino acid sequence alignments of PYMV DNA ORF-encoded proteins with those encoded by other previously sequenced geminivirus ORFs show that PYMV is closely related to those geminiviruses isolated from the New World, especially in the putative coat protein gene regions.

Characterization of potato yellow mosaic virus as a geminivirus with a bipartite genome.

A whitefly-transmitted virus from Venezuela, potato yellow mosaic virus, has been propagated, isolated and characterized as a member of the Geminivirus group. The virus was transmitted to several species of Nicotiana and to Petunia hybrida by mechanical inoculation, and to potato and tomato plants by grafting. Purified virus possessed typical geminate particle morphology and encapsidated both genomic and subgenomic species consisting of single-stranded DNA. The genome of the virus was cloned into both bacteriophage and plasmid vectors following restriction of supercoiled double-stranded DNA species isolated from infected plant extracts. Two distinct classes of cloned DNA were generated, both about 2,500-2,600 base pairs in length and designated A and B, which when inoculated in combination to Nicotiana benthamiana were infectious after excision from the recombinant clones. The subgenomic DNA was shown to be related to DNA B.